Conventional methods for the detection of disease in the commercial fish production industry is limited to bacterial cell culture and biochemical testing following natural outbreaks of disease. This slow and reactive process is inadequate to protect this valuable industry from the economic losses that can result from infectious diseases of fish.
While efforts have been made in the development of real-time PCR assays for the detection of particular pathogens in plants and humans, none have been developed to assist in disease prevention and control in commercial catfish farms. The present invention involves the development of a real-time PCR assay for detection of the leading pathogen affecting commercial catfish farms in the southeastern United States.
Enteric septicemia of catfish (ESC) is among the most prevalent and costly disease affecting channel catfish (Ictalurus punctatus) commercial production. The causative agent of ESC is the gram-negative bacterium Edwardsiella ictaluri (Hawke, et al., 1981). Pathogen uptake occurs primarily through the gut, nares, and gills (Baldwin and Newton, 1993) and appears to be facilitated by active feeding during periods of disease outbreak (Wise and Johnson, 1998). Progression of ESC is usually rapid, most significantly affecting fingerlings (Francis-Floyd, et al., 1987), with clinical disease signs manifesting within a few days post-infection followed by death usually within one to two weeks (Wise, et al., 1997). Resistant fish may harbor the pathogenic bacteria in macrophages, however it is unclear what role the engulfed bacteria play in later outbreaks of infection (Shoemaker and Klesius, 1997). Edwardsiella ictaluri is known to persist in pond sediments for at least one month (Plumb and Quinlan, 1986). While the pathology of the disease is well documented (Shotts, et al., 1986; Newton, et al., 1989; Baldwin and Newton, 1993), a better understanding of the rate of pathogen clearance would improve efforts in selective breeding for disease resistance and assist management of disease outbreaks in farm populations by providing methods of evaluating transmission rates occurring in natural outbreaks.
Currently, bacterial cell culture and biochemical testing are the primary means of detecting Edwardsiella ictaluri in natural outbreaks. Tissue samples are cultured for 48 hours on blood-heart infusion agar (BHI) followed by biochemical analysis to confirm putative bacterial infection. By this time, fish are typically exhibiting external signs of infection and the spread and progression of the disease throughout the population is likely (Klesius, 1994). Sensitivity of culturing assays is relatively poor, resulting in reliable detection of the bacterium only at moderate to high concentrations. Other more rapid methods (FAT and ELISA) have been developed for ESC detection (Rogers, 1981; Klesius, et al., 1991), but also lack reliability at low levels of infection (Nogva and Lillehaug, 1999; Nogva, et al., 2000).
Detection assays using PCR-based technologies have been developed for a variety of pathogens that occur in fish, soil, and other systems (Makino, et al., 1993; Leon, et al., 1994; Leal-Klevezas, et al., 1995; Mariam, et al., 1997; Nogva and Lillehaug, 1999; Bell, et al., 1999; Nogva, et al., 2000; Vishnubhatla, et al., 2000; Kimura, et al., 2001). These assays typically provide greater levels of sensitivity than traditional diagnostic tools (bacterial cell culture and characterization, immunological, and serological assays). Until recently, accurate quantitative assessment of pathogen levels has been problematic when infection levels are low.
A need therefore exists for an early, fast, effective method of detecting the causative agent, Edwardsiella ictaluri, of Enteric Septicemia of catfish (ESC).